A successful infection involves virus entry into the cell; uncoating, expression, and replication of the genome; assembly and release of infectious virus particles; and defense against specific and non-specific host immune mechanisms. Combined genetic, biochemical, electron microscopic, and immunologic approaches are being used to investigate these complex processes. During the past year we continued work on the assembly of vaccinia virus. We identified a protein component of the intracellular mature vaccinia virion membrane that is a target of a potent neutralizing monoclonal antibody. By immunofluorescent and electron microscopic analysis, the antibody was found to stain intracytoplasmic viral factories, virion membranes in cell sections and the surface of negatively stained purified virions. The antigen, which is synthesized at late times in infection, has apparent molecular masses of 25 kDa and 29 kDa under non-reducing and reducing conditions, respectively. Although the N-terminus of the immunoaffinity purified protein was blocked, sequence analysis of trypic peptides revealed that the antigen was identical to the myristylated protein encoded by the LIR open reading frame. Validation of this genetic assignment was provided by the ability of the antibody to immunoprecipitate a [3H]myristic acid-labeled product of the expected molecular weight from infected cells. We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profilin, and characterized it. The viral profilin differed from the vertebrate homolog by having a higher affinity for polyphosphoinositides than actin suggesting that it may have a primary role in phosphoinositide metablolism.